Environmental stress-responsive promoter and genes encoding transcriptional factor

ABSTRACT

The present invention provides a stress responsive promoter. The environmental stress responsive promoter of the present invention comprises DNA of the following (a), (b) or (c):(a) DNA consisting of any nucleotide sequence selected from SEQ ID NOS: 1 to 90; (b) DNA consisting of a nucleotide sequence comprising a deletion, substitution or addition of one or more nucleotides relative to any nucleotide sequence selected from SEQ ID NOS: 1 to 90, and functioning as an environmental stress responsive promoter; and (c) DNA hybridizing under stringent conditions to DNA consisting of any nucleotide sequence selected from SEQ ID NOS: 1 to 90, and functioning as an environmental stress responsive promoter.

TECHNICAL FIELD

The present invention relates to an environmental stress-responsivepromoter and a gene encoding environmental stress-responsivetranscriptional factor.

BACKGROUND ART

Large quantities of genomic and cDNA sequences have been determined withrespect to a number of organisms by gene sequencing projects. In a plantmodel, Arabidopsis thaliana, the complete genomic sequences of twochromosomes have been determined (Lin, X. et al., (1999), Nature 402,761-768; and Mayer, K. et al., (1999), Nature 402, 769-777).

The expressed sequence tag (EST) project also has greatly contributed tothe discovery of expression genes (Hofte, H. et al., (1993), Plant J. 4,1051-1061; Newman, T. et al., (1994), Plant Physiol. 106, 1241-1255; andCooke, R. et al., (1996), Plant J. 9, 101-124; and Asamizu, E. et al.,(2000), DNA Res. 7, 175-180). For example, the database of EST (dbEST)of the National Center for Biotechnology Information(NCBI) includespartial cDNA sequences, in which more than half (about 28,000 genes) ofthe total genes are reproduced, (as estimated from the gene content ofArabidopsis thaliana chromosome 2 completely sequenced [Lin, X. et al.,(1999), Nature 402, 761-768]).

Recently, microarray (DNA chip) technology has become a useful tool foranalyzing genome-scale gene expression (Schena, M. et al., (1995),Science 270, 467-470; Eisen, M. B. and Brown, P. O. (1999), MethodsEnzymol. 303, 179-205). In the technology using a DNA chip, cDNAsequences are arrayed on a slide glass in a density of not smaller than1,000 genes/cm ². The cDNA sequences thus arrayed are hybridizedsimultaneously with a pair of cDNA probes tagged with two colorfluorescent labels, which have been prepared from RNA samples ofdifferent types of cells or tissues. In this manner, a large amount ofgenes can be directly analyzed and compared for gene expression. Thistechnology was demonstrated for the first time by analyzing 48Arabidopsis genes for differential expression in root and shoots(Schena, M. et al., (1995), Science 270, 467-470). Furthermore, amicroarray was used in investigating 1,000 clones randomly taken from ahuman cDNA library in order to identify a novel gene responsive to heatshock and protein kinase C activation (Schena, M. et al., (1996), Proc.Natl. Acad. Sci. USA, 93, 10614-10619).

In another method, a DNA chip is used in analyzing the expressionprofile of an inflammatory-disease associated gene under variousinduction conditions (Heller, R. A. et al., (1997), Proc. Natl. Acad.Sci. USA, 94, 2150-2155). Furthermore, using a microarray, a yeastgenome having more than 6,000 coding sequences has been analyzed fordynamic expression (DeRisi, J. L. et al., (1997) Science 278, 680-686;and Wodicka, L. et al., (1997), Nature Biotechnol. 15, 1359-1367).

However, in the field of plant science, only a few reports have beenmade on microarray analysis (Schena, M. et al., (1995), Science 270,467-470; Ruan, Y. et al., (1998), Plant J. 15, 821-833; Aharoni. A. etal., (2000), Plant Cell 12, 647-661; and Reymond, P. et al., (2000),Plant Cell 12, 707-719).

The growth of plants is significantly affected by environmental stressessuch as drought, high salinity and low temperature. Of the stresses,drought or water deficiency is the most critical factor that limitsgrowth of plants and production of crops. Such a drought stress causesvarious biochemical and physiological responses in plants.

To survive under these conditions of stress, plants acquire responsivityand adaptability to the stresses. Recently, several types of genesresponsive to drought at a transcriptional level have been reported(Bohnert, H. J. et al., (1995), Plant Cell 7, 1099-1111; Ingram, J., andBartels, D. (1996), Plant Mol. Biol. 47, 377-403; Bray, E. A. (1997),Trends Plant Sci. 2, 48-54; Shinozaki, K., and Yamaguchi-Shinozaki, K.(1997), Plant Physiol. 115, 327-334; Shinozaki, K., andYamaguchi-Shinozaki, K. (1999), “Molecular responses to drought stress.Molecular responses to cold, drought, heat and salt stress in higherplants”, edited by Shinozaki, K. and Yamaguchi-Shinozaki, K. R. G.Landes Company; and Shinozaki, K., and Yamaguchi-Shinozaki, K. (2000),Curr. Opin. Plant Biol. 3, 217-223).

On the other hand, in an attempt to improve stress resistance of plantsby introducing a gene, stress-inducible genes have been used (Holmberg,N., and Bulow, L. (1998), Trends Plant Sci. 3, 61-66; and Bajaj, S. etal., (1999), Mol. Breed. 5, 493-503). Not only to further clarify themechanism of stress resistance and stress responsivity of a higher plantat a molecular level but also to improve the stress resistance of a cropby gene manipulation, it is important to analyze the function of astress-inducible gene.

Dehydration responsive element and C-repeat sequence (DRE/CRT) has beenidentified as an important cis-acting element when drought, high saltand cold stress-responsive genes are expressed in an ABA independentmanner, where ABA refers to abscisic acid, a kind of plant hormone andserves as a signal transmission factor of seed dormancy andenvironmental stress (Yamaguchi-Shinozaki, K., and Shinozaki, K. (1994),Plant Cell 6, 251-264; Thomashow, M. F. et al., (1999), Plant Mol. Biol.50, 571-599; and Shinozaki, K., and Yamaguchi-Shinozaki, K. (2000),Curr. Opin. Plant Biol. 3, 217-223). Furthermore, a transcriptionalfactor (DREB/CBF) involved in DRE/CRT responsive gene expression hasbeen cloned (Stockinger. E. J. et al., (1997), Proc. Natl. Acad. Sci.USA 94, 1035-1040; Liu, Q. et al., (1998), Plant Cell 10, 1391-1406;Shinwari, Z. K. et al., (1998), Biochem. Biophys. Res. Commun. 250,161-170; and Gilmour, S. J. et al., (1998), Plant J. 16, 433-443).DREB1/CBF is considered to function in cold-responsive gene expression,whereas DREB2 is involved in drought-responsive gene expression. Strongresistance to freezing stress was observed in a transgenic Arabidopisplant that overexpresses CBF1 (DREB1B) CDNA under the control of acauliflower mosaic virus (CaMV) 35S promoter (Jaglo-Ottosen, K. R. etal., (1998), Science 280, 104-106).

The present inventors have reported that when DREB1A (CBF3) cDNAmolecules are overexpressed in transgenic plants under the control of aCaMV 35S promoter or a stress-inducible rd29A promoter, strongconstitutive expression of stress-inducible DREB1A target genes areinduced to improve resistance to freezing, drought and salt stresses(Liu, Q. et al., (1998), Plant Cell 10, 1391-1406; and Kasuga, M. etal., (1999), Nature Biotechnol. 17, 287-291). Furthermore, the presentinventors have already identified six DREB1A target genes such asrd29A/lti78/cor78, kin1, kin2/cor6.6, cor15a, rd17/cor47, and erd10(Kasuga, M. et al., (1999), Nature Biotechnol. 17, 287-291). However, ithas not yet been sufficiently elucidated how the overexpressed DREB1AcDNA improves stress resistance to freezing, drought and salt in atransgenic plant. To investigate the molecular mechanisms of drought andfreezing resistance, it is important to identify and analyze as manygenes controlled by DREB1A as possible.

DISCLASURE OF THE INVENTION

The present invention is directed to providing an environmentalstress-responsive promoter and a gene encoding an environmentalstress-responsive transcriptional factor.

The present inventors have intensively studied to solve theaforementioned problems. As a result, they succeeded in identifyingnovel genes responsive to cold, drought and salt stresses and isolatingpromoter regions thereof by using CDNA microarray analysis, therebyaccomplishing the present invention.

More specifically, the present invention is directed to an environmentalstress-responsive promoter comprising DNA of the following (a), (b) or(c):

-   (a) DNA consisting of any nucleotide sequence selected from SEQ ID    NOS: 1 to 90;-   (b) DNA consisting of a nucleotide sequence comprising a deletion,    substitution or addition of one or more nucleotides relative to any    nucleotide sequence selected from SEQ ID NOS: 1 to 90, and    functioning as an environmental stress responsive promoter; and-   (c) DNA hybridizing under stringent conditions to DNA consisting of    any nucleotide sequence selected from SEQ ID NOS: 1 to 90, and    functioning as an environmental stress responsive promoter.

Examples of environmental stress include at least one selected from thegroup consisting of cold stress, drought stress, and salt stress.

The present invention is also directed to an expression vectorcomprising the promoter mentioned above, or an expression vector havingan arbitrary gene integrated therein.

Furthermore, the present invention is directed to a transformantcomprising the expression vector.

Moreover, the present invention is directed to a transgenic plant, suchas a plant body, plant organ, plant tissue or plant culture cell,comprising the expression vector.

The present invention is still further directed to a method forproducing a stress-resistant plant, comprising culturing or cultivatingthe transgenic plant.

On the other hand, the present inventors identified novel genes encodingcold, drought and salt stress-responsive transcriptional factors by useof cDNA microarray analysis, thereby accomplishing the presentinvention.

More specifically, the present invention is directed to a gene encodingan environmental stress-responsive transcriptional factor comprising anamino acid of the following (a) or (b):

-   (a) any amino acid sequence selected from SEQ ID NOS: 2n (n is an    integer from 47 to 82);-   (b) an amino acid sequence comprising a deletion, substitution or    addition of one or more amino acids relative to any amino acid    sequence selected from SEQ ID NOS: 2n (n is an integer from 47 to    82), functioning as an environmental stress-responsive    transcriptional factor.

Also, the present invention is directed to a gene according to claim 1,comprising DNA of the following (a), (b) or (c):

-   (a) DNA consisting of any nucleotide sequence selected from SEQ ID    NOS: 2n−1 (n is an integer from 47 to 82);-   (b) DNA consisting of a nucleotide sequence comprising a deletion,    substitution or addition of one or more nucleotides relative to any    nucleotide sequence selected from SEQ ID NOS: 2n−1 (n is an integer    from 47 to 82), and encoding an environmental stress-responsive    transcriptional factor; and-   (c) DNA hybridizing under stringent conditions to DNA consisting of    any nucleotide sequence selected from SEQ ID NOS: 2n−1 (n is an    integer from 47 to 82), and encoding an environmental    stress-responsive transcriptional factor.

In the present invention, examples of environmental stress include atleast one selected from the group consisting of cold stress, droughtstress, and salt stress.

The present invention is also directed to an expression vectorcontaining the gene, a transformant containing the expression vector,and a transgenic plant containing the expression vector.

Furthermore, the present invention is directed to a transgenic plant,such as a plant body, plant organ, plant tissue or plant culture cell.

Moreover, the present invention is directed to a method for producing astress-resistant plant, comprising culturing or cultivating thetransgenic plant.

Hereinafter, the present invention will be described in detail.

The present inventors constructed full-length cDNA libraries fromArabidopsis plants placed under different conditions, such asdehydration-treated plants and cold-treated plants (Seki. M. et al.,(1998), Plant J. 15, 707-720), by the biotinylated CAP trapper method(Carninci. P. et al., (1996), Genomics, 37, 327-336). Then, Arabidopsisfull-length cDNA microarrays were respectively prepared using about1,300 full-length cDNA molecules and about 7,000 full-length cDNAmolecules both containing stress-inducible genes. Besides using thesedehydration and cold-inducible full-length cDNA molecules, another cDNAmicroarray was prepared using a DREB1A target gene, a transcriptionalregulator for controlling expression of a stress-responsive gene.Thereafter, expression patterns of genes under drought and cold stresswere monitored to exhaustively analyze stress-responsive genes. As aresult, from the full-length cDNA microarray containing about 1,300 offull-length cDNA molecules, novel environmental stress-responsive genes,that is, 44 drought-inducible genes and 19 cold-inducible genes wereisolated. 30 out of the 44 drought-inducible genes, and 10 out of the 19cold-inducible genes were novel stress-inducible genes. Moreover, it wasfound that 12 stress-inducible genes were DREB1A target genes and 6 outof the 12 stress-inducible genes were novel genes. As a result of theanalysis, 301 drought-inducible genes, 54 cold-inducible genes and 211high salt-stress inducible genes were isolated from a cDNA microarraycontaining about 7,000 full-length cDNA molecules.

Thereafter, not only promoter regions but also environmental genesencoding environmental stress-responsive transcriptional factors weresuccessfully isolated from these environmental stress-responsive genes.

As described above, a full-length cDNA microarray is useful tool foranalyzing the expression manner of Arabidopsis thaliana drought- andcold-stress inducible genes and analyzing the target gene of a stressassociated transcriptional regulator.

1. Isolation of Promoter

The promoter of the present invention contains a cis-element which ispresent upstream of a gene encoding a stress-responsive proteinexpressed by an environmental stress such as a cold, drought, or highsalt stress and which activates the transcription of a gene presentdownstream thereof by binding of a transcriptional factor. Examples ofsuch a cis-element include a dehydration responsive element (DRE), anabscisic acid responsive element (ABRE), and a cold-stress responsiveelement. Examples of genes encoding proteins binding to these elementsinclude a DRE binding protein 1A gene (referred to also as a “DREB1Agene”), DRE binding protein 1C gene (referred to also as a “DREB1Cgene”), DRE binding protein 2A gene (referred to also as a “DREB2Agene”), and DRE binding protein 2B gene (referred to also as a “DREB2Bgene”).

In isolating a promoter of the present invention, first,stress-responsive genes are isolated by using a microarray. Inconstructing a microarray, use may be made of about 1,300 cDNA moleculesin total including genes isolated from Arabidopsis full-length cDNAlibraries, responsive to dehydration (RD) genes, early responsive todehydration (ERD) genes, kin1 genes, kin2 genes, and cor15a genes; andfurthermore, α-tubulin genes as an internal standard; and moreover,mouse nicotinic acetylcholine receptor epsilon subunit (nAChRE) genesand mouse glucocorticoid receptor homologous genes, as negativecontrols.

As a microarray used in isolating the promoter of the present invention,use may be made of about 7,000 cDNA molecules in total including genesisolated from an Arabidopsis full-length cDNA library, responsive todehydration (RD) genes, early responsive to dehydration(ERD) genes, andPCR amplification fragments as an internal standard obtained from λcontrol template DNA fragments (TX803, manufactured by Takara Shuzo);and mouse nicotinic acetylcholine receptor epsilon subunit (nAChRE)genes and mouse glucocorticoid receptor homologous genes, as negativecontrols.

A plasmid DNA extracted with a plasmid preparation device (manufacturedby Kurabo) is sequenced by sequence analysis using a DNA sequencer (ABIPRISM 3700, PE Applied Biosystems, CA, USA). Based on the GenBank/EMBLdatabase, the obtained sequence is screened for homology by using theBLAST program.

After poly A selection is performed, reverse transcription is carriedout to synthesize double-stranded DNA molecules and a cDNA molecule isinserted into a vector.

The cDNA molecule inserted into a vector for constructing cDNA librariesis amplified by PCR using complementary primers to the sequences ofvectors on both sides of the cDNA molecule. Examples of such vectorsinclude λZAPII and λPS.

A microarray can be prepared according to a conventional method, whichis not particularly limited. For example, using a gene tip microarraystamp machine GTMASS SYSTEM (manufactured by Nippon Laser & ElectronicsLab.), the above obtained PCR product is loaded from a microtiter plateand spotted on a microslide glass at predetermined intervals. Then, toprevent a non-specific signal from being expressed, the slide isimmersed into a blocking solution.

Examples of plant materials include a plant strain obtained bydestroying specific genes as well as wild type plants. A transgenicplant having cDNA of DREB1A introduced therein may be used. Examples ofplant species include Arabidopsis thaliana, tobacco and rice. Of them,Arabidopsis thaliana is preferable.

Dehydration- and cold-stress treatments can be carried out according toa known method (Yamaguchi-Shinozaki, K., and Shinozaki, K. (1994), PlantCell 6, 251-264).

After plant bodies (wild type plants and DREB1A overexpressiontransformants) are exposed to stress, they are sampled and stored incryogenic conditions with liquid nitrogen. The wild type and DREB1Aoverexpression transformants are used in an experiment to identify aDREB1A target gene. From plant bodies, mRNA is isolated and purified bya known method or a kit.

In the presence of Cy3 dUTP or Cy5 dUTP for labeling (AmershamPharmacia), each of mRNA samples is subjected to reverse transcriptionand then used in hybridization.

After the hybridization, the microarray is scanned with a scanning lasermicroscope or the like. As a program for analyzing data of a microarray,Imagene Ver 2.0 (BioDiscovery) and QuantArray (GSI Lumonics) etc. may beused.

After the scanning, a plasmid having a target gene is prepared. In thisway, the target genes are isolated.

A promoter region is determined by analyzing the nucleotide sequence ofthe gene isolated above and using a gene analysis program based on thegenomic information of database (GenBank/EMBL, ABRC). The isolated genescan be classified into ones inducible by both dehydration and coldstress, ones inducible specifically by drought stress, and one induciblespecifically by cold stress. According to the gene analysis program,from the genes mentioned above, 90 types of genes below can beidentified.

(FL03-07-F12, FL04-12-F24, FL04-14-N10, FL04-14-P24, FL04-17-I03,FL04-17-M08, FL04-17-M22, FL05-05-A17, FL05-05-F20, FL05-05-G20,FL05-09-N09, FL05-10-J09, FL05-10-M08, FL05-11-H09, FL05-12-H13,FL05-13-I20, FL05-14-E15, FL05-14-E16, FL05-16-F03, FL05-16-H23,FL05-18-M07, FL05-18-O21, FL05-19-F21, FL05-19-O22, FL05-21-K17,FL06-10-F03, FL06-12-H12, FL07-12-I23, FL08-08-H23, FL08-08-O14,FL08-09-M05, FL08-10-K08, FL08-11-P07, FL08-13-F10, FL08-19-D04,FL08-19-G15, FL09-06-B11, FL09-07-G17, FL09-10-A12, FL09-13-P15,FL02-05-I05, FL04-12-N15, FL04-16-P21, FL04-17-N22, FL04-20-P19,FL02-09-H01, FL05-01-D08, FL05-02-G08, FL05-02-O17, FL05-07-L13,FL05-08-B14, FL05-09-N10, FL05-11-L01, FL05-12-J09, FL05-14-D24,FL05-14-F20, FL05-14-I08, FL05-15-C04, FL05-15-E19, FL05-18-A06,FL05-18-H15, FL05-19-C02, FL05-20-M16, FL05-20-N18, FL05-21-E06,FL05-21-L12, FL06-07-B08, FL06-08-H20, FL06-09-N04, FL06-11-K21,FL07-07-G15, FL07-12-D17, FL08-11-C23, FL08-13-G20, FL08-15-M21,FL08-18-N19, FL08-19-C07, FL08-19-P05, FL09-07-G09, FL09-07-G15,FL09-10-J18, FL09-11-I12, FL09-12-B03, FL09-16-I11, FL09-16-M04,FL11-01-J18, FL11-07-D13, FL11-07-F02, FL11-07-N15 and FL11-10-D10). Thepromoter regions of these genes are represented by SEQ ID NOS: 1 to 90,respectively.

As long as a promoter of the present invention acts as an environmentalstress-responsive promoter, use may be made of any promoter having anucleotide sequence selected from SEQ ID NOS: 1 to 90 wherein one ormore nucleotides, preferably one or several nucleotides (for example 1to 10, preferably 1 to 5) may be deleted, substituted or added.Furthermore, DNA hybridizing with the DNA comprising any nucleotidesequence selected from SEQ ID NOS: 1 to 90 under stringent conditionsand acting as an environmental stress-responsive promoter is alsoincluded in the promoter of the present invention.

Once the nucleotide sequence of a promoter according to the presentinvention is determined, the promoter can be obtained by chemicalsynthesis, PCR using a cloned probe as a template, or hybridizationusing a DNA fragment having the nucleotide sequence as a probe.Furthermore, a mutant of the promoter of the present invention, whichhas the same functions as those of a non-mutated promoter, can be alsosynthesized by a site-specific mutagenesis or the like.

To introduce a mutation into a promoter sequence, a known method such asthe Kunkel method, Gapped duplex method or an equivalent method may beemployed. A mutation may be introduced by using a mutation-introducingkit (for example, Mutant-K manufactured by Takara or Mutant-Gmanufactured by Takara) which uses a site-specific mutagenesis or byusing the LA PCR in vitro mutagenesis series kit (manufactured byTakara).

The term “functioning as an environmental stress-responsive promoter”used herein refers to a function of activating transcription caused bybinding RNA polymerase to the promoter when the promoter is exposed to apredetermined environmental stress condition.

The term “environmental stress” used herein generally refers to anabiotic stress such as drought stress, cold stress, high salt stress, orintensive light stress. The term “drought” used herein refers to a stateof water deficiency, and the term “cold” used herein refers to a statewhere an object is exposed to a lower temperature than the optimumliving temperature for each organism (e.g., in the case of Arabidopsisthaliana, it is exposed to a temperature of −20 to +21° C. continuouslyfor one hour to several weeks). The term “high salt” used herein refersto a state where a plant is treated with NaCl of 50 mM to 600 mM inconcentration continuously for 0.5 hours to several weeks. The term“intensive light stress” used herein refers to a state where toointensive light to use for photosynthesis is applied to a plant, andcorresponds to a case where, for example, light of 5,000 to 10,000 Lx ormore is applied. These environmental stresses may be applied singly orin combination.

The plant promoter of the present invention includes a promoter having anucleotide sequence represented by SEQ ID NOS: 1 to 90 wherein anucleotide sequence may be added to the 3′ end in order to increasetranscriptional efficiency or a nucleotide sequence may be deleted fromthe 5′ end to the extent not to lose the activity of a promoter.

Furthermore, the promoter of the present invention includes DNA whichhybridizes with DNA consisting of any nucleotide sequence selected fromSEQ ID NOS: 1 to 90 under stringent conditions and acts as anenvironmental stress-responsive promoter. The term “stringentconditions” used herein refers to the conditions of sodium concentrationof 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68°C., preferably 42 to 65° C.; more preferably, conditions of 5×SSC (83 mMNaCl, 83 mM sodium citrate) and a temperature of 42° C.

2. Construction of Expression Vector

An expression vector of the present invention can be obtained byligating (inserting) a promoter according to the present invention to anappropriate vector. The vector into which a promoter of the presentinvention is to be inserted is not particularly limited as long as itcan be replicated in a host. Examples of such a vector include aplasmid, shuttle vector and helper plasmid.

Examples of such a plasmid DNA include plasmids derived from Escherichiacoli (e.g., pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, andpBluescript); plasmids derived from Bacillus subtilis (e.g., pUB110 andpTP5); and plasmids derived from yeasts (e.g., YEp13 and YCp50).Examples of a phage DNA include λ phages (Charon4A, Charon21A EMBL3,EMBL4, λgt10, λgt11, and λZAP). Further animal virus vectors such asretrovirus and a vaccinia virus and insect virus vectors such as abaculovirus can be also used.

To insert a promoter according to the present invention into a vector,use may be made of a method of digesting a purified DNA with appropriaterestriction enzymes, inserting the obtained DNA fragment into therestriction site of a suitable vector DNA or a multi-cloning site, andligating it to the vector.

In the present invention, to express an arbitrary gene, the arbitrarygene can be further inserted into the aforementioned expression vector.The technique inserting an arbitrary gene is the same as the methodinserting a promoter into a vector. An arbitrary gene is notparticularly limited. Examples of the gene include genes shown in Table2 and known genes other than those.

In a case where a reporter gene, for example, a GUS gene, widely used inplants is linked to the 3′ end of a promoter of the present invention,the strength of the promoter can be easily evaluated by checking GUSactivity. As such a reporter gene other than the GUS gene, luciferaseand a green fluorescent protein can be used.

As described above, various types of vectors can be used in the presentinvention. Further, a desired gene is ligated to the promoter of thepresent invention in a sense or antisense direction and then, theconstruction can be inserted into a vector such as pBI101 (Clonetech)called a binary vector.

3. Isolation of Transcriptional Factor

A transcriptional factor binds to a cis element which is presentupstream of a gene and activates the transcription of the gene presentdownstream thereof. The transcriptional factors isolated in the presentinvention are induced by environmental stresses such as a lowtemperature, dehydration, and high salt concentration.

Environmental stress-responsive transcriptional factors are roughlydivided into those belonging to a DREB family, ERF family, zinc fingerfamily, WRKY family, MYB family, bHLH family, NAC family, homeo domainfamily and bZIP family.

In isolating a transcriptional factor, first, stress responsive genesare isolated by using a microarray. As a microarray, use may be made ofabout 7,000 cDNA molecules in total including genes isolated fromArabidopsis full-length cDNA libraries, responsive to dehydration (RD)genes, early responsive to dehydration (ERD) genes; PCR amplificationfragments obtained from a λ control template DNA fragment (TX803,manufactured by Takara Shuzo), as an internal standard; and mousenicotinic acetylcholine receptor epsilon subunit (nAChRE) genes andmouse glucocorticoid receptor homologous genes, as negative controls.

A plasmid DNA extracted by a plasmid preparation device (manufactured byKurabo) is sequenced by sequence analysis using a DNA sequencer (ABIPRISM 3700, PE Applied Biosystems, CA, USA). Based on the GenBank/EMBLdatabase, the obtained sequence is screened for homology by using theBLAST program.

After poly A selection is performed, reverse transcription is carriedout to synthesize a double-stranded DNA molecule and a cDNA molecule isinserted into a vector.

The cDNA molecule inserted into a vector for constructing cDNA librariesis amplified by PCR using complementary primers to the sequences ofvectors on both sides of the cDNA molecule. Examples of such vectorsinclude λZAPII and λPS.

A microarray can be prepared according to a conventional method, whichis not particularly limited. For example, using a gene tip microarraystamp machine GTMASS SYSTEM (manufactured by Nippon Laser & ElectronicsLab.), the above obtained PCR product is loaded from the microtiterplate and spotted on a microslide glass at predetermined intervals.Then, to prevent a non-specific signal from being expressed, the slideis immersed into a blocking solution.

Examples of plant materials include a plant strain obtained bydestroying a specific gene as well as wild type plants. A transgenicplant having a cDNA of DREB1A introduced therein may be used. Examplesof plant species include Arabidopsis thaliana, tobacco and rice. Ofthem, Arabidopsis thaliana is preferable.

Dehydration- and cold-stress treatments can be carried out according toa known method (Yamaguchi-Shinozaki, K., and Shinozaki, K. (1994), PlantCell 6, 251-264).

After plant bodies (wild type plants and DREB1A overexpressiontransformants) are exposed to stress, they are sampled and stored incryogenic conditions with liquid nitrogen. The wild type and DREB1Aoverexpression transformants are used in an experiment to identify aDREB1A target gene. From plant bodies, mRNA is isolated and purified bya known method or a kit.

In the presence of Cy3 dUTP or Cy5 dUTP for labeling (AmershamPharmacia), each of mRNA samples is subjected to reverse transcriptionand then used in hybridization.

After hybridization, the microarray is scanned with a scanning lasermicroscope or the like. As a program for analyzing data of a microarray,Imagene Ver 2.0 (BioDiscovery) and QuantArray (GSI Lumonics) etc., maybe used.

After the scanning, a plasmid having a target gene is prepared. In thisway, the target genes are isolated.

A transcriptional factor is determined by analyzing the nucleotidesequence of the gene isolated above and using a gene analysis programbased on the genomic information of database (GenBank/EMBL, ABRC). Theisolated genes can be classified into ones inducible by both drought andcold stress, ones inducible specifically by drought stress, and oneinducible specifically by cold stress. According to the gene analysisprogram, from the genes mentioned above, genes encoding 36 types oftranscriptional factors can be identified. The nucleotide sequences ofthe genes encoding 36 types of transcriptional factors are representedby SEQ ID NOS: 2n−1 (n is an integer of 47 to 82) and amino acidsequences of the transcriptional factors are represented by SEQ ID NOS:2n (n is an integer of 47 to 82). Sequence ID numbers and the names ofgenes encoding transcriptional factors are shown in Table 1. TABLE 1Name of gene SEQ ID NO: RAFL05-11-M11 SEQ ID NO: 93 RAFL06-11-K21 SEQ IDNO: 95 RAFL05-16-H23 SEQ ID NO: 97 RAFL08-16-D06 SEQ ID NO: 99RAFL08-16-G17 SEQ ID NO: 101 RAFL06-08-H20 SEQ ID NO: 103 RAFL07-10-G04SEQ ID NO: 105 RAFL04-17-D16 SEQ ID NO: 107 RAFL05-19-M20 SEQ ID NO: 109RAFL08-11-M13 SEQ ID NO: 111 RAFL04-15-K19 SEQ ID NO: 113 RAFL05-11-L01SEQ ID NO: 115 RAFL05-14-C11 SEQ ID NO: 117 RAFL05-19-G24 SEQ ID NO: 119RAFL05-20-N02 SEQ ID NO: 121 RAFL05-18-H12 SEQ ID NO: 123 RAFL06-10-D22SEQ ID NO: 127 RAFL06-12-M01 SEQ ID NO: 129 RAFL05-14-D24 SEQ ID NO: 131RAFL05-20-N17 SEQ ID NO: 133 RAFL04-17-F21 SEQ ID NO: 135 RAFL09-12-N16SEQ ID NO: 137 RAFL05-19-I05 SEQ ID NO: 139 RAFL05-21-I22 SEQ ID NO: 141RAFL08-11-H20 SEQ ID NO: 143 RAFL05-21-C17 SEQ ID NO: 145 RAFL05-08-D06SEQ ID NO: 147 RAFL05-20-M16 SEQ ID NO: 149 RAFL11-01-J18 SEQ ID NO: 151RAFL11-09-C20 SEQ ID NO: 153 RAFL05-18-N16 SEQ ID NO: 155 RAFL11-10-D10SEQ ID NO: 157 RAFL04-17-N22 SEQ ID NO: 159 RAFL05-09-G15 SEQ ID NO: 161RAFL05-21-L12 SEQ ID NO: 163

Note that as long as a transcriptional factor of the present inventionfunctions as an environmental stress-responsive transcriptional factor,use may be made of any transcriptional factor having a nucleotidesequence selected from SEQ ID NOS: 2n−1 (n is an integer of 47 to 82)wherein one or more nucleotides, preferably one or several nucleotides(for example 1 to 10, preferably 1 to 5) have been deleted, substitutedor added. Furthermore, DNA hybridizing with the DNA comprising anynucleotide sequence selected from SEQ. ID NOS. 2n−1 (n is an integer of47 to 82) under stringent conditions and encoding an environmentalstress-responsive transcriptional factor is also included in thetranscriptional factor of the present invention. The term “stringentconditions” used herein refers to the conditions of sodium concentrationof 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 42 to 68°C., preferably 42 to 65° C.; more preferably, conditions of 5×SSC (83 mMNaCl, 83 mM sodium citrate) and a temperature of 42° C.

36 types of transcriptional factors isolated in the present inventionmay be classified as follows.

-   (1) DREB family: RAFL05-11-M11, RAFL06-11-K21, RAFL05-16-H23,    RAFL08-16-D16;-   (2) ERF family: RAFL08-16-G17, RAFL06-08-H20;-   (3) Zinc finger family: RAFL07-10-G04, RAFL04-17-D16, RAFL05-19-M20,    RAFL08-11-M13, RAFL04-15-K19, RAFL05-11-L01, RAFL05-14-C11,    RAFL05-19-G24, RAFL05-20-N02;-   (4) WRKY family: RAFL05-18-H12, RAFL05-19-E19, RAFL06-10-D22,    RAFL06-12-M01;-   (5) MYB family: RAFL05-14-D24, RAFL05-20-N17, RAFL04-17-F21;-   (6) bHLH family: RAFL09-12-N16;-   (7) NAC family: RAFL05-19-I05, RAFL05-21-I22, RAFL08-11-H20,    RAFL05-21-C17, RAFL05-08-D06;-   (8) Homeo domain family: RAFL05-20-M16, RAFL11-01-J18,    RAFL11-09-C20; and-   (9) bZIP family: RAFL05-18-N16, RAFL11-10-D10, RAFL04-17-N22,    RAFL05-09-G15.

Note that RAFL05-21-L12 cannot be classified into (1) to (9).

Once the nucleotide sequence of a gene encoding a transcriptional factoraccording to the present invention is determined, the gene encoding atranscriptional factor according to the present invention can beobtained by chemical synthesis, PCR using a cloned probe as a template,or hybridizing a DNA fragment having the nucleotide sequence as a probe.Furthermore, a mutant of the gene encoding a transcriptional factoraccording to the present invention, and having the same functions asthose of a non-mutated transcriptional factor, can be also synthesizedby a site-specific mutagenesis or the like.

To introduce a mutation into a nucleotide sequence of a gene encoding atranscriptional factor, a known method such as the Kunkel method, Gappedduplex method, or an equivalent method may be employed. A mutation maybe introduced by using a mutation-introducing kit (for example, Mutant-Kmanufactured by Takara and Mutant-G manufactured by Takara) which uses asite-specific mutagenesis or by using the LA PCR in vitro mutagenesisseries kit (manufactured by Takara).

The term “environmental stress” used herein generally refers to anabiotic stress such as drought stress, cold stress, high salt stress, orintensive light stress. The term “drought” used herein refers to a stateof water deficiency, the term “cold” used herein refers to a state wherean object is exposed to a lower temperature than the optimum livingtemperature of each organism (e.g., in the case of Arabidopsis thaliana,e.g., in the case of Arabidopsis thaliana, it is exposed to atemperature of −20 to +21° C. continuously for one hour to severalweeks). The term “high salt” used herein refers to a state where a plantis treated with NaCl of 50 mM to 600 mM in concentration continuouslyfor 0.5 hours to several weeks. The term “intensive light stress” usedherein refers to a state where too intensive light to use forphotosynthesis is applied to a plant, and corresponds to a case where,for example, light of 5,000 to 10,000 Lx or more is applied. Theseenvironmental stresses may be applied singly or in combination.

4. Construction of Expression Vector

The expression vector of the present invention can be obtained byligating (inserting) a gene encoding a transcriptional factor accordingto the present invention to an appropriate vector. The vector into whicha gene encoding a transcriptional factor of the present invention isinserted is not particularly limited as long as it can be replicated ina host. Examples of such a vector include a plasmid, shuttle vector andhelper plasmid.

Examples of such a plasmid DNA include plasmids derived from Escherichiacoli (e.g., pBR322, pBR325, pUC118, pUC119, pUC18, pUC19, andpBluescript), plasmids derived from Bacillus subtilis (e.g., pUB110 andpTP5); and plasmids derived from yeasts (e.g., YEp13 and YCp50).Examples of a phage DNA include λ phages (Charon4A, Charon21A EMBL3,EMBL4, λgt10, λgt11, and λZAP). Further animal virus vectors such asretrovirus and a vaccinia virus and insect virus vectors such as abaculovirus can be also used.

To insert a transcriptional factor of the present invention into avector, use may be made of a method of digesting a purified DNA withappropriate restriction enzymes, inserting the obtained DNA fragmentinto the restriction site of a suitable vector DNA or a multi-cloningsite, and ligating it to the vector.

In a case where a reporter gene, for example, a GUS gene, widely used inplants is linked to the 3′ end of the gene encoding a transcriptionalfactor of the present invention, the strength of the gene expression canbe easily evaluated by checking GUS activity. As such a reporter geneother than the GUS gene, luciferase and a green fluorescent protein canbe used.

5. Preparation of Transformant

A transformant of the present invention can be obtained by introducingan expression vector of the present invention into a host. The host usedherein is not particularly limited as long as it can express a promoter,a gene of interest, or an environmental stress-responsivetranscriptional factor. Of them, a plant is preferable. In a case of aplant host, a transformant plant (transgenic plant) can be obtained asfollows.

A plant to be transformed in the present invention refers to an entireplant, a plant organ (such as leaf, petal, stem, root, or seed), a planttissue (such as the epidermis, phloem, parenchyma, xylem, or vascularbundle), or a plant culture cell. Examples of plants used fortransformation include plants belonging to the Brassicaceae, Graniineae,Solanaceae and Leguminosae (see below); however they are not limited tothese plants.

Brassicaceae: Arabidopsis thaliana

Gramineae: Nicotiana tabacum

Solanaceae: Zea mays, Oryza sativa

Leguminosae: Glycine max

The aforementioned recombinant vector can be introduced into a plant bya conventional transformation method such as electroporation,Agrobacterium method, particle gun method, or PEG method.

For example, where electroporation is used, a gene is introduced into ahost by treating a vector by an electroporation device equipped with apulse controller under conditions: a voltage of 500 to 1,600 V, 25 to1,000 μF, and 20 to 30 msec.

When a particle gun method is used, a plant body, organ and tissue maybe directly used. Alternatively, they may be used after they aresectioned to pieces or after protoplasts of them are prepared. Thesamples thus prepared may be processed by a gene-introduction device(for example, PDS-1000/He manufactured by Bio-Rad). Processingconditions vary depending upon a plant or sample. Generally, processingis performed at a pressure of about 1,000 to 1800 psi and a distance ofabout 5 to 6 cm.

Furthermore, a gene of interest can be introduced into a plant by usinga plant virus as a vector. Examples of available plant viruses include acauliflower mosaic virus. More specifically, a virus genome is insertedinto a vector derived from Escherichia coli to prepare a recombinant andthen such a gene of interest is inserted into the virus genome. Thevirus genome thus modified is excised out from the recombinant withrestriction enzymes and inoculated into a plant host. In this manner thegene of interest can be introduced into the plant host.

In the method using a Ti plasmid of the Agrobacterium, when bacteriabelonging to the Agrobacterium are transfected to a plant, a portion ofplasmid DNA of the bacteria is transferred into a plant genome. Usingsuch a characteristic, a gene of interest is introduced into a planthost. Of bacteria belonging to the Agrobacterium, Agrobacteriumtumefaciens, when it is introduced into a plant by transfection,produces a tumor called a crown gall. Also, a plant when it istransfected with Agrobacterium rhizogenes, it produces hairy roots.These phenomena are caused by transferring a region called a T-DNAregion (transferred DNA region) present in a plasmid such as a Tiplasmid or Ri plasmid present in each bacterium into a plant andincorporating the region into a plant genome at a time of transfection.

By inserting desired DNA, which is to be incorporated into a plantgenome, into the T-DNA region on a Ti or Ri plasmid, the desired DNA canbe incorporated into a plant genome, when the host is transfected withAgrobacterium bacteria.

Tumoral tissues, shoots and hairy roots obtained as a result oftransformation can be directly used in cell culture, tissue culture, ororgan culture. Also, when a plant hormone such as auxin, cytokinin,gibberellin, abscisic acid, ethylene, or brassinoride, is administeredto them in an appropriate concentration by using a conventional planttissue culture method, a plant body can be regenerated from them.

A vector according to the present invention can be not only incorporatedinto the plant hosts mentioned above but also introduced into bacteriabelonging to the Escherichia such as Escherichia coli, the Bacillus suchas Bacillus subtilis and the Pseudomonas such as Pseudomonas putida;yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe;animal cells such as COS cells and CHO cells; and insect cells such asSf9 cells, to obtain a transformant. Where a bacterium such asEscherichia coli or yeast is used as a host, it is preferable that arecombinant vector according to the present invention can beself-replicated in the bacterium and, at the same time, is comprised ofa promoter of the present invention, a ribosome binding sequence, a geneof interest and a transcription termination sequence. Furthermore, agene regulating the promoter may be included in the bacterium.

A method for introducing a recombinant vector into bacteria is notparticularly limited as long as it is a method which can introduce DNAinto bacteria. Examples of such a method include a method of usingcalcium ions and an electroporation method.

When a yeast is used as a host, Saccharomyces cerevisiae andSchizosaccharomyces pombe may be used. A method for introducing arecombinant vector is not particularly limited as long as it is a methodfor introducing DNA into a yeast. Examples of such a method includeelectroporation, spheroplast method, and lithium acetate method.

Where an animal cell is used as a host, a monkey COS-7 cell, Vero,Chinese hamster ovary cell (CHO cell), and mouse L cell etc. are used.Examples of methods for introducing a recombinant vector into an animalcell include electroporation, calcium phosphate method, and lipofectionmethod.

When an insect cell is used as a host, a Sf9 cell and the like may beused. Examples of method for introducing a recombinant vector into aninsect cell include a calcium phosphate method, lipofection method, andelectroporation method.

Whether a gene is incorporated into a host or not is confirmed by a PCRmethod, Southern hybridization, Northern hybridization method or thelike. For example, PCR is performed by preparing DNA from atransformant, and designing DNA specific primers. PCR is carried outunder the same conditions as used for preparing the plasmid mentionedabove. Thereafter, the obtained amplified product is subjected toagarose gel electrophoresis, polyacrylamide gel electrophoresis orcapillary electrophoresis, and stained with ethidium bromide, or SYBRGreen solution, etc. If the amplified product is found as a single band,it is confined that a transformant is obtained. Alternatively, theamplified product can be also detected by PCR using primers previouslystained with a fluorescent dye or the like. Furthermore, there may beemployed a method in which the amplified product is bound to a solidphase such as a microplate and confirmed by fluorescent or an enzymaticreaction.

4. Production of Plant

In the present invention, a transformed plant body can be regeneratedfrom the above transformed plant cell or the like. As a regenerationmethod, use is made of one in which callus-form transformed cells aretransferred to a medium having a different hormone in a differentconcentration and cultured to form an adventitious embryo, from which anentire plant body is obtained. Examples of the medium to be used hereininclude an LS medium and an MS medium.

The “method for producing a plant body” of the present inventioncomprises steps of: introducing a plant expression vector, into whichthe above plant promoter or a gene encoding an environmentalstress-responsive transcriptional factor is inserted, into a host cellto obtain a transformed plant cell; regenerating a transformed plantbody from the transformed plant cell; obtaining a plant seed from thetransformed plant body; and producing a plant body from the plant seed.

To obtain plant seeds from a transformed plant body, for example, thetransformed plant body is collected from a rooting medium andtransferred to a pot having soil containing water placed therein. Then,the transformed plant body is grown at constant temperature to formflowers. Finally seeds are obtained. To produce a plant body from aseed, for example, when a seed formed on a transformed plant body hasmatured, the seed is isolated and seeded in soil containing water,followed by growing at constant temperature under illumination. Theplant thus bred becomes an environmental stress-resistant plantexhibiting the stress resistance corresponding to the responsivity of apromoter introduced therein or a gene encoding the environmentalstress-responsive transcriptional factor introduced therein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL03-07-F12;

FIG. 2 is a characteristic graph showing the relationship between coldtreatment time and expression ratio regarding FL04-12-F24;

FIG. 3 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL04-14-N10;

FIG. 4 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL04-14-P24;

FIG. 5 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL04-17-I03;

FIG. 6 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL04-17-I03;

FIG. 7 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL04-17-M08;

FIG. 8 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL04-17-M22;

FIG. 9 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-05-A17;

FIG. 10 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-05-F20;

FIG. 11 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-05-G20;

FIG. 12 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-09-N09;

FIG. 13 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-10-J09;

FIG. 14 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-10-J09;

FIG. 15 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-10-M08;

FIG. 16 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-11-H09;

FIG. 17 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-12-H13;

FIG. 18 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-12-H13;

FIG. 19 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL05-13-I20;

FIG. 20 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-14-E15;

FIG. 21 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-14-E16;

FIG. 22 is a characteristic graph showing the relationship between coldtreatment time and expression ratio regarding FL05-14-E16;

FIG. 23 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL05-14-E16;

FIG. 24 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-16-F03;

FIG. 25 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL05-16-F03;

FIG. 26 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-16-H23;

FIG. 27 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-16-H23;

FIG. 28 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-18-M07;

FIG. 29 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL05-18-M07;

FIG. 30 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL05-18-021;

FIG. 31 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-19-F21;

FIG. 32 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL05-19-F21;

FIG. 33 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-19-O22;

FIG. 34 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-19-O22;

FIG. 35 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL05-19-O22;

FIG. 36 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-21-K17;

FIG. 37 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL06-10-F03;

FIG. 38 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL06-12-H12;

FIG. 39 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL06-12-H12;

FIG. 40 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL07-12-I23;

FIG. 41 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL08-08-H23;

FIG. 42 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-08-O14;

FIG. 43 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-09-M05;

FIG. 44 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL08-10-K08;

FIG. 45 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-11-P07;

FIG. 46 is a characteristic graph showing the relationship between coldtreatment time and expression ratio regarding FL08-11-P07;

FIG. 47 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-13-F10;

FIG. 48 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL08-13-F10;

FIG. 49 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL08-13-F10;

FIG. 50 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-19-D04;

FIG. 51 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL08-19-G15;

FIG. 52 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL09-06-B11;

FIG. 53 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL09-07-G17;

FIG. 54 is a characteristic graph showing the relationship between ABAtreatment time and expression ratio regarding FL09-10-A12;

FIG. 55 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL09-13-P15;

FIG. 56 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL02-05-I05;

FIG. 57 is a characteristic graph showing the relationship between coldtreatment time and expression ratio regarding FL04-12-N15;

FIG. 58 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL04-16-P21;

FIG. 59 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL04-17-N22;

FIG. 60 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL04-20-P19;

FIG. 61 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL02-09-H01;

FIG. 62 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-01-D08;

FIG. 63 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-02-G08;

FIG. 64 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-02-O17;

FIG. 65 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-07-L13;

FIG. 66 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-08-B14;

FIG. 67 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-09-N10;

FIG. 68 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-11-L01;

FIG. 69 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-12-J09;

FIG. 70 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-14-D24;

FIG. 71 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-14-F20;

FIG. 72 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-14-I08;

FIG. 73 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-15-C04;

FIG. 74 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-15-E19;

FIG. 75 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-18-A06;

FIG. 76 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL05-18-H 15;

FIG. 77 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-19-C02;

FIG. 78 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-20-M16;

FIG. 79 is a characteristic graph showing the relationship between coldtreatment time and expression ratio regarding FL05-20-N18;

FIG. 80 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-21-E06;

FIG. 81 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL05-21-L12;

FIG. 82 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL06-07-B08;

FIG. 83 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL06-08-H20;

FIG. 84 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL06-09-N04;

FIG. 85 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL06-11-K21;

FIG. 86 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL07-07-G15;

FIG. 87 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL07-12-D17;

FIG. 88 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-11-C23;

FIG. 89 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-13-G20;

FIG. 90 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-15-M21;

FIG. 91 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-18-N19;

FIG. 92 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL08-19-C07;

FIG. 93 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL08-19-P05;

FIG. 94 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL09-07-G09;

FIG. 95 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL09-07-G15;

FIG. 96 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL09-10-J18;

FIG. 97 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL09-11-I12;

FIG. 98 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL09-12-B03;

FIG. 99 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL09-16-I11;

FIG. 100 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL09-16-M04;

FIG. 101 is a characteristic graph showing the relationship betweendehydration treatment time and expression ratio regarding FL11-01-J18;

FIG. 102 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL11-07-D13;

FIG. 103 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL11-07-F02;

FIG. 104 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL11-07-N15;

FIG. 105 is a characteristic graph showing the relationship between highsalt treatment time and expression ratio regarding FL11-10-D10;

FIG. 106 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL08-16-G17;

FIG. 107 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-11-M11;

FIG. 108 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-11-M11;

FIG. 109 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL06-11-K21;

FIG. 110 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL06-11-K21;

FIG. 111 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL06-08-H20;

FIG. 112 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL06-08-H20;

FIG. 113 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-16-H23;

FIG. 114 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-16-H23;

FIG. 115 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL08-16-D06;

FIG. 116 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL07-10-G04;

FIG. 117 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL04-17-D16;

FIG. 118 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-19-M20;

FIG. 119 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL08-11-M13;

FIG. 120 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL04-15-K19;

FIG. 121 is a characteristic graph showing the relationship between coldstress and expression ratio regarding RAFL04-15-K19;

FIG. 122 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-11-L01;

FIG. 123 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-11-L01;

FIG. 124 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-14-C11;

FIG. 125 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-19-G24;

FIG. 126 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-19-G24;

FIG. 127 is a characteristic graph showing the relationship between coldstress and expression ratio regarding RAFL05-19-G24;

FIG. 128 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-20-N02;

FIG. 129 is a, characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-18-H12;

FIG. 130 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-18-H12;

FIG. 131 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-19-E19;

FIG. 132 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL06-10-D22;

FIG. 133 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL06-12-M01;

FIG. 134 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL06-12-M01;

FIG. 135 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-14-D24;

FIG. 136 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-14-D24;

FIG. 137 is a characteristic graph showing the relationship between coldstress and expression ratio regarding RAFL05-20-N17;

FIG. 138 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-20-N17;

FIG. 139 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL04-17-F21;

FIG. 140 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL09-12-N16;

FIG. 141 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding AFL05-19-I05;

FIG. 142 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-19-I05;

FIG. 143 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-21-I22;

FIG. 144 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL08-11-H20;

FIG. 145 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL08-11-H20;

FIG. 146 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-21-C17;

FIG. 147 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-21-C17;

FIG. 148 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-08-D06;

FIG. 149 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-20-M16;

FIG. 150 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-20-M16;

FIG. 151 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL11-01-J18;

FIG. 152 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL11-01-J18;

FIG. 153 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL11-09-C20;

FIG. 154 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-18-N16;

FIG. 155 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL11-10-D10;

FIG. 156 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL11-10-D10;

FIG. 157 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL04-17-N22;

FIG. 158 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL04-17-N22;

FIG. 159 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-09-G15;

FIG. 160 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-09-G15;

FIG. 161 is a characteristic graph showing the relationship betweendrought stress and expression ratio regarding RAFL05-21-L12; and

FIG. 162 is a characteristic graph showing the relationship between highsalt stress and expression ratio regarding RAFL05-21-L12.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be further explained in detailby way of examples, which should not be construed as limiting the scopeof the present invention.

EXAMPLE 1. Isolation of Promoter

1. Materials and Methods

(1) Arabidopsis cDNA Clone

A microarray was constructed by using about 7,000 cDNA molecules intotal including genes isolated from an Arabidopsis full-length cDNAlibraries, responsive-to-dehydration (RD) genes, earlyresponsive-to-dehydration (ERD) genes, kin 1 genes, kin2 genes, andcor15a genes; α-tubulin genes as an internal standard; and mousenicotinic acetylcholine receptor epsilon subunit (nAChRE) genes andmouse glucocorticoid receptor homologous genes, as negative controls.

-   Positive control: dehydration-inducible genes    (responsive-to-dehydration genes: rd, and early    responsive-to-dehydration genes: erd)-   Internal standard: α-tubulin gene-   Negative control: mouse nicotinic acetylcholine receptor epsilon    subunit (nAChRE) genes and mouse glucocorticoid receptor homologous    genes, which do not substantially have homology with any given    sequence in an Arabidopsis database for analyzing non-specific    hybridization.    (2) Arabidopsis Full-Length cDNA Microarray

The present inventors have constructed full-length cDNA libraries froman Arabidopsis plant body under different conditions (e.g., dehydrationtreatment, cold treatment and non-treatment in different growth stagesfrom budding to maturation of seeds) by the biotinylated CAP trappermethod. From the full-length cDNA libraries, the present inventorsisolated individually about 7,000 independent Arabidopsis full-lengthcDNA molecules. The cDNA fragments, which were amplified by PCR, werearranged on a slide glass in accordance with a known method (Eisen andBrown, 1999). The present inventors prepared a full-length cDNAmicroarray containing about 7,000 Arabidopsis full-length cDNAmolecules, which contain the genes below.

(3) Isolation of Dehydration-, Cold-, High Salt-, and ABA-InducibleGenes Using cDNA Microarray

In this example, dehydration-, cold-, high salt-, and ABA-induciblegenes were isolated by using a full-length cDNA microarray containingabout 7,000 Arabidopsis full-length cDNA molecules.

Probes of a plant treated with different stresses and an untreated plantwith stress and labeled with Cy3 and Cy5 fluorescent dyes were mixed.The probes were hybridized with the full-length cDNA microarraycontaining about 7,000 Arabidopsis full-length cDNA molecules. By such adouble labeling of a pair of cDNA probes wherein one of the mRNA sampleswas labeled with Cy3-dUTP and the other was labeled with Cy5-dUTP,hybridization with DNA elements on a microarray can be performedsimultaneously, with the result that quantitative determination of geneexpression under two different conditions (that is, stressed andunstressed conditions) can be directly and easily performed. Thehybridized microarray was scanned by two discrete laser channels for Cy3and Cy5 emission from each of DNA elements. Subsequently, the intensityratio between two fluorescent signals from each DNA element wasdetermined. Based on the relative value of the intensity ratio, a changeof differential expression of genes represented as a cDNA spot on themicroarray was determined. In this example, an α-tubulin gene, whoseexpression level was almost equivalent under two different experimentalconditions was used, as an internal control gene.

In the full-length cDNA microarray containing about 7,000 Arabidopsisfull-length cDNA molecules, a procedure for identifying dehydration-,cold-, high salt-, and ABA-inducible genes will be explained.

-   1) Both mRNA molecules derived from a plant treated with one of the    stresses mentioned above and mRNA molecules derived from a wild-type    plant unstressed were used to prepare Cy3-labeled cDNA and    Cy5-labeled cDNA probes, respectively. These cDNA probes were mixed    and hybridized with the cDNA microarray. In this example, an    α-tubulin gene, which exhibits almost the same expression level    under two type conditions, was as used as an internal control gene.    A gene that exhibits the expression ratio of dehydration:unstressed,    cold:unstressed, or high salt:unstressed more than double of that of    the α-tubulin gene was defined as an inducible gene by a stress    given to the gene.-   2) Both mRNA molecules derived from a 35S:DREB1A transgenic plant    and mRNA molecules derived from a wild-type plant unstressed were    used to prepare Cy3-labeled cDNA and Cy5-labeled cDNA probes,    respectively. These cDNA probes were mixed and hybridized with a    cDNA microarray. In this example, an α-tubulin gene exhibiting    almost the same expression level under two type conditions was used    as an internal control gene. A gene of 35S:DREB1A transgenic plant    exhibiting an expression ratio more than double of that of a gene of    the wild type plant unstressed was defined as a DREB1A target gene.

Both mRNA molecules derived from a plant treated with a stress and mRNAmolecules derived from a wild-type plant unstressed were used to prepareCy3-labeled cDNA and Cy5-labeled cDNA probes, respectively. These cDNAprobes were mixed and hybridized with a cDNA microarray. The sameexperiment was repeated three times to evaluate the reproducibility ofmicroarray analysis. When the same mRNA sample was hybridized withvarious microarrays, a good correlation was observed. A gene thatexhibits an expression ratio (dehydration/unstressed, cold/unstressed)more than double of that of the α-tubulin gene was defined as aninducible gene by a stress given to the gene.

(4) Analysis of Sequence

Plasmid DNA extracted by a plasmid preparation device (NA 100)manufactured by Kurabo was sequenced to find homology of gene sequences.The DNA sequence was determined by a dye terminator cycle sequencingmethod using a DNA sequencer (ABI PRISM 3700. PE Applied Biosystems, CA,USA). Based on the GenBank/EMBL database, homology of sequences wasfound by using the BLAST program.

(5) Amplification of cDNA

λZAPII (Carninci et al., 1996) was used as a vector for constructing acDNA library. The cDNA inserted in a vector for the library wasamplified by PCR using complementary primers to the sequences of bothsides of the cDNA.

The sequences of primers are as follows: FL forward 1224:5′-CGCCAGGGTTTTCCCAGTCACGA (SEQ ID NO: 91) FL reverse 1233:5′-AGCGGATAACAATTTCACACAGGA (SEQ ID NO: 92)

To 100 μl of a PCR solution mixture (0.25 mM dNTP, 0.2 μM PCR primer,1×Ex Taq Buffer, and 1.25 U Ex Taq polymerase (manufactured by TakaraShuzo)), a plasmid (1 to 2 ng) was added as a template. PCR wasperformed under the following conditions: an initial reaction at 94° C.for 3 minutes, 35 cycles each consisting of 95° C. for one minute, 60°C. for 30 seconds and 72° C. for 3 minutes, and a final reaction at 72°C. for 3 minutes. After a PCR product was precipitated with ethanol, theprecipitate was dissolved in 25 μl of 3×SSC and then subjected toelectrophoresis using 0.7% agarose gel. The quality of the DNA obtainedand amplification efficiency of PCR were confirmed.

(6) Construction of cDNA Microarray

Using a gene tip microarray stamp machine GTMASS SYSTEM (manufactured byNippon Laser & Electronics Lab.), 0.5 μl of a PCR product (100 to 500ng/ml) was loaded from a 384-well microtiter plate to form spots of thePCR product (5 nl for each) at intervals of 280 μm on 6 micro slideglasses (S7444, manufactured Matsunami) coated with poly-L lysine. Tospot DNA in an equivalent amount, the slide after printing was placed ina beaker containing heated distilled water to moisten it and placed at100° C. for 3 seconds to dry it. After the slide was placed on a sliderack, the rack was transferred into a glass chamber. To the glasschamber, a blocking solution (15 ml of 1M sodium borate salt (pH 8.0),5.5 g succinic anhydrous compound (Wako), and 335 ml of1-methyl-2-pyrrolidon (Wako)) was poured. After the glass chamberhousing the slide rack was shaken up and down 5 times and gently shakingfor 15 minutes, the slide rack was transferred to a glass chambercontaining boiling water, shaken 5 times, and allowed to stand alone for2 minutes. Thereafter, the slide rack was transferred to a glass chambercontaining 95% ethanol, shaken 5 times, and centrifuged at 800 rpm for30minutes.

(7) Plant Material and Isolation of RNA

As a plant material, use was made of a wild type Arabidopsis thalianaplant body which was seeded on an agar medium and grown for 3 weeks(Yamaguchi-Shinozaki and Shinozaki, 1994) and an Arabidopsis thaliana(Colombian species) plant body into which DREB1A cDNA(Kasuga et al.,1999) connected to a 35S promoter of a cauliflower mosaic virus wasintroduced. Dehydration- and cold-stress treatments were performed inaccordance with the method of Yamaguchi-Shinozaki and Shinozaki (1994).More specifically, dehydration treatment was performed by pulling aplant body out of the agar medium, placing it on a filter, and dried ata temperature of 22° C. and a relative humidity of 60%. The coldtreatment was performed by transferring a plant body grown at 22° C. to4° C. High salt stress treatment was performed by growing a plant bodyat an aqueous solution containing 250 mM NaCl.

After wild type plant bodies were exposed to stress-treatment for 2 or10 hours, a sample was taken from each of plant bodies and stored incryogenic conditions with liquid nitrogen. Furthermore, wild type andDREB1A overexpresstion-type transformants cultured in an agar mediumwithout kanamycin were subjected to an experiment for identifying aDREB1A target gene. The DREB1A overexpresstion-type transformant was nottreated with stresses. The total RNA was isolated from a plant body byusing ISOGEN (Nippon gene, Tokyo, Japan) and mRNA was isolated andpurified by Oligotex-dT30 mRNA purification kit (Takara, Tokyo, Japan).

(8) Fluorescent Labeling of Probe

Each of the mRNA samples was subjected to a reverse transcriptionreaction in the presence of Cy3 dUTP or Cy5 dUTP (Amersham Pharmacia).The composition of the buffer (30 μl) used in the reverse transcriptionreaction is shown in Table 2. TABLE 2 poly(A)⁺ RNA with 6 μg oligo(dT)18-mer  1 μg 10 mM DTT 500 μM dATP, dCTP and dGTP 200 μM dTTP 100 μM Cy3dUTP or Cy5 dUTP 400 units of SuperScript II Reverse Transcriptase (Lifetechnologies) 1X Superscript First Strand Synthesis Buffer (Lifetechnologies) Total 30 μL

After reaction was performed at 42° C. for one hour, two samples(labeled with Cy3 and Cy5) were mixed to obtain a reaction mixture. Tothis reaction mixture, 15 μl of 0.1 M NaOH and 1.5 μl of 20 mM EDTA wereadded and treated at 70° C. for 10 minutes. Further, 15 μl of 0.1 M HClwas added to the reaction mixture, a sample was taken and transferred toa Micro con 30 micro concentrator (Amicon). 400 μl of TE buffer wasadded to the sample and centrifuged until the volume of the bufferreached 10 to 20 μl. The effulent was discarded. 400 μl of TE buffer and20 μl of 1 mg/ml human Cot-1 DNA (Gibco BRL) were added to the resultantmixture and the mixture was again centrifuged. The labeled samples werecentrifugally collected and several μl of distilled water was addedthereto. The obtained probes, 2 μl of 10 μg/μl yeast tRNA, 2 μl of 1μg/μl pd(A)₁₂₋₁₈ (Amersham Pharmacia), 3.4 ml of 20×SSC, and 0.6 μl of10% SDS were added. Further, the samples were denatured at 100° C. for 1minute and placed at room temperature for 30 minutes and thereafter usedin hybridization.

(9) Microarray Hybridization and Scanning

A probe was subjected to high-speed centrifugation for one minute by abenchtop micro centrifuge. To avoid generation of bubbles, the probe wasplaced at the center of an array and a cover slip was placed thereon.Four drops of 5 μl of 3×SSC were dropped on a slide glass and a chamberwas kept at a suitable humidity to prevent the probe from being driedduring hybridization. After the slide glass was placed in a cassette forhybridization (THC-1, BM machine) and the cassette was sealed,hybridization treatment was performed at 65° C. for 12 to 16 hours. Theslide glass was taken out from the cassette and placed on the sliderack. After the cover slip was carefully removed in solution 1 (2×SSC,0.1% SDS), the rack was washed while shaking and transferred intosolution 2 (1×SSC) to wash for 2 minutes. The rack was furthertransferred to solution 3 (0.2×SSC), allowed to stand for 2 minutes, andcentrifuged at 800 rpm for 1 min to dry.

The microarray was scanned at a resolution of 10 μm per pixel by use ofa scanning laser microscope (ScanArray 4000; GSI Lumonics, Watertown,Mass.). As a program for analyzing microarray data, Imagene Ver 2.0(BioDiscovery) and QuantArray (GSI Lumonics) were used.

(10) Northern Analysis

Northern analysis was performed using total RNA, (Yamaguchi-Shinozakiand Shinozaki, 1994). DNA fragments were isolated from the Arabidopsisthaliana full-length cDNA library by a PCR method and used as probes forNorthern hybridization.

(11) Determination of Promoter Region

Based on the genomic information of Arabidopsis thaliana in a data base(GenBank/EMBL, ABRC), a promoter region was analyzed by using the BLASTprogram for gene analysis.

2. Results

(1) Stress-Inducible Gene

Fluorescent-labeled cDNA was prepared by subjecting mRNA isolated froman unstressed Arabidopsis thaliana plant to a reverse transcriptionreaction in the presence of Cy5-dUTP. A second probe labeled withCy3-dUTP was prepared from a plant treated at low temperature for 2hours. Both probes were simultaneously hybridized with a cDNA microarraycomprising about 7,000 Arabidopsis thaliana cDNA clones and then apseudo color image was created.

Genes induced and suppressed by a stress are represented by a red signaland green signal, respectively. Genes expressed at virtually the samelevel in both treatments are represented by a yellow signal. Theintensity of each spot corresponds to the absolute value of theexpression level of each gene. It is shown that a cold-inducible gene(rd29A) is represented by a red signal whereas an α-tubulin gene (aninternal control) is represented by a yellow signal.

(2) Identification of Promoter Region

As a result of identifying a promoter region, the promoter gene regionsof 90 types of genes were obtained in a full-length cDNA microarraycontaining about 7,000 of Arabidopsis full-length cDNA molecules. Thename of these 90 types of genes and their promoter sequences aresummarized in Table 3 TABLE 3 Name of gene SEQ ID NO: FL03-07-F12 SEQ IDNO: 1 FL04-12-F24 SEQ ID NO: 2 FL04-14-N10 SEQ ID NO: 3 FL04-14-P24 SEQID NO: 4 FL04-17-I03 SEQ ID NO: 5 FL04-17-M08 SEQ ID NO: 6 FL04-17-M22SEQ ID NO: 7 FL05-05-A17 SEQ ID NO: 8 FL05-05-F20 SEQ ID NO: 9FL05-05-G20 SEQ ID NO: 10 FL05-09-N09 SEQ ID NO: 11 FL05-10-J09 SEQ IDNO: 12 FL05-10-M08 SEQ ID NO: 13 FL05-11-H09 SEQ ID NO: 14 FL05-12-H13SEQ ID NO: 15 FL05-13-I20 SEQ ID NO: 16 FL05-14-E15 SEQ ID NO: 17FL05-14-E16 SEQ ID NO: 18 FL05-16-F03 SEQ ID NO: 19 FL05-16-H23 SEQ IDNO: 20 FL05-18-M07 SEQ ID NO: 21 FL05-18-O21 SEQ ID NO: 22 FL05-19-F21SEQ ID NO: 23 FL05-19-O22 SEQ ID NO: 24 FL05-21-K17 SEQ ID NO: 25FL06-10-F03 SEQ ID NO: 26 FL06-12-H12 SEQ ID NO: 27 FL07-12-I23 SEQ IDNO: 28 FL08-08-H23 SEQ ID NO: 29 FL08-08-O14 SEQ ID NO: 30 FL08-09-M05SEQ ID NO: 31 FL08-10-K08 SEQ ID NO: 32 FL08-11-P07 SEQ ID NO: 33FL08-13-F10 SEQ ID NO: 34 FL08-19-D04 SEQ ID NO: 35 FL08-19-G15 SEQ IDNO: 36 FL09-06-B11 SEQ ID NO: 37 FL09-07-G17 SEQ ID NO: 38 FL09-10-A12SEQ ID NO: 39 FL09-13-P15 SEQ ID NO: 40 FL02-05-I05 SEQ ID NO: 41FL04-12-N15 SEQ ID NO: 42 FL04-16-P21 SEQ ID NO: 43 FL04-17-N22 SEQ IDNO: 44 FL04-20-P19 SEQ ID NO: 45 FL02-09-H01 SEQ ID NO: 46 FL05-01-D08SEQ ID NO: 47 FL05-02-G08 SEQ ID NO: 48 FL05-02-O17 SEQ ID NO: 49FL05-07-L13 SEQ ID NO: 50 FL05-08-B14 SEQ ID NO: 51 FL05-09-N10 SEQ IDNO: 52 FL05-11-L01 SEQ ID NO: 53 FL05-12-J09 SEQ ID NO: 54 FL05-14-D24SEQ ID NO: 55 FL05-14-F20 SEQ ID NO: 56 FL05-14-I08 SEQ ID NO: 57FL05-15-C04 SEQ ID NO: 58 FL05-15-E19 SEQ ID NO: 59 FL05-18-A06 SEQ IDNO: 60 FL05-18-H15 SEQ ID NO: 61 FL05-19-C02 SEQ ID NO: 62 FL05-20-M16SEQ ID NO: 63 FL05-20-N18 SEQ ID NO: 64 FL05-21-E06 SEQ ID NO: 65F105-21-L12 SEQ ID NO: 66 FL06-07-B08 SEQ ID NO: 67 FL06-08-H20 SEQ IDNO: 68 FL06-09-N04 SEQ ID NO: 69 FL06-11-K21 SEQ ID NO: 70 FL07-07-G15SEQ ID NO: 71 FL07-12-D17 SEQ ID NO: 72 FL08-11-C23 SEQ ID NO: 73FL08-13-G20 SEQ ID NO: 74 FL08-15-M21 SEQ ID NO: 75 FL08-18-N19 SEQ IDNO: 76 FL08-19-C07 SEQ ID NO: 77 FL08-19-P05 SEQ ID NO: 78 FL09-07-G09SEQ ID NO: 79 FL09-07-G15 SEQ ID NO: 80 FL09-10-J18 SEQ ID NO: 81FL09-11-I12 SEQ ID NO: 82 FL09-12-B03 SEQ ID NO: 83 FL09-16-I11 SEQ IDNO: 84 FL09-16-M04 SEQ ID NO: 85 FL11-01-J18 SEQ ID NO: 86 FL11-07-D13SEQ ID NO: 87 FL11-07-F02 SEQ ID NO: 88 FL11-07-N15 SEQ ID NO: 89FL11-10-D10 SEQ ID NO: 90(3) The Relationship between Stress Treatment Time and Expression Ratio

The 90 types of stress inducible genes isolated above were analyzed forthe relationship between stress treatment time and expression ratio. Theresults are shown in FIGS. 1 to 105. The relationship between 90 typesof genes and stress treatment are shown in Table 4. TABLE 4 Name of geneType of applied stress Drawing FL03-07-F12 Dehydration FL04-12-F24Exposure to cold FL04-14-N10 Dehydration FL04-14-P24 DehydrationFL04-17-I03 Dehydration, Exposure to a high level salt solutionFL04-17-M08 Exposure to a high level salt solution FL04-17-M22Dehydration FL05-05-A17 Dehydration FL05-05-F20 Dehydration FL05-05-G20Dehydration FL05-09-N09 Dehydration FL05-10-J09 Dehydration, Exposure toa high level salt solution FL05-10-M08 Exposure to a high level saltsolution FL05-11-H09 Exposure to a high level salt solution FL05-12-H13Dehydration, Exposure to a high level salt solution FL05-13-I20 ABAtreatment FL05-14-E15 Dehydration FL05-14-E16 Dehydration, Exposure tocold, ABA treatment FIGS. 21-23 FL05-16-F03 Dehydration, ABA treatmentFL05-16-H23 Dehydration, Exposure to a high level salt solutionFL05-18-M07 Dehydration, ABA treatment FL05-18-O21 ABA treatmentFL05-19-F21 Dehydration, ABA treatment FL05-19-O22 Dehydration, Exposureto a high level salt solution, ABA FIGS. 33-35 treatment FL05-21-K17Exposure to a high level salt solution FL06-10-F03 ABA treatmentFL06-12-H12 Dehydration, Exposure to a high level salt solutionFL07-12-I23 Exposure to a high level salt solution FL08-08-H23 Exposureto a high level salt solution FL08-08-O14 Dehydration FL08-09-M05Dehydration FL08-10-K08 Exposure to a high level salt solutionFL08-11-P07 Dehydration, Exposure to cold FL08-13-F10 Dehydration,Exposure to a high level salt solution, ABA FIGS. 47-49 treatmentFL08-19-D04 Dehydration FL08-19-G15 Exposure to a high level saltsolution FL09-06-B11 ABA treatment FL09-07-G17 ABA treatment FL09-10-A12ABA treatment FL09-13-P15 Dehydration FL02-05-I05 Exposure to a highlevel salt solution FL04-12-N15 Exposure to cold FL04-16-P21 DehydrationFL04-17-N22 Exposure to a high level salt solution FL04-20-P19Dehydration FL02-09-H01 Dehydration FL05-01-D08 Dehydration FL05-02-G08Exposure to a high level salt solution FL05-02-O17 DehydrationFL05-07-L13 Exposure to a high level salt solution FL05-08-B14Dehydration FL05-09-N10 Dehydration FL05-11-L01 Dehydration FL05-12-J09Dehydration FL05-14-D24 Dehydration FL05-14-F20 Dehydration FL05-14-I08Dehydration FL05-15-C04 Dehydration FL05-15-E19 Dehydration FL05-18-A06Dehydration FL05-18-H15 Exposure to a high level salt solutionFL05-19-C02 Dehydration FL05-20-M16 Dehydration FL05-20-N18 Exposure tocold FL05-21-E06 Dehydration FL05-21-L12 Dehydration FL06-07-B08Dehydration FL06-08-H20 Dehydration FL06-09-N04 Dehydration FL06-11-K21Dehydration FL07-07-G15 Exposure to a high level salt solutionFL07-12-D17 Exposure to a high level salt solution FL08-11-C23Dehydration FL08-13-G20 Dehydration FL08-15-M21 Dehydration FL08-18-N19Dehydration FL08-19-C07 Dehydration FL08-19-P05 Exposure to a high levelsalt solution FL09-07-G09 Exposure to a high level salt solutionFL09-07-G15 Dehydration FL09-10-J18 Exposure to a high level saltsolution FL09-11-I12 Dehydration FL09-12-B03 Dehydration FL09-16-I11Exposure to a high level salt solution FL09-16-M04 Exposure to a highlevel salt solution FL11-01-J18 Dehydration FL11-07-D13 Exposure to ahigh level salt solution FL11-07-F02 Exposure to a high level saltsolution FL11-07-N15 Exposure to a high level salt solution FL11-10-D10Exposure to a high level salt solution

In FIGS. 1 to 105, the vertical axis shows the expression ratio of agene, which is calculated as follows:Expression ratio=[(FI of a cDNA molecule under stress)/(FI of a cDNAmolecule under no stress)]÷[(FI of α-tubulin under stress)/(FI ofα-tubulin under no stress)]where FI is the intensity of fluorescence.

As shown in FIGS. 1 to 105, the stress inducible genes isolated by amethod according to the present invention exhibit different profiles;however, it is found that expression is induced by adding each stress.From this, it is demonstrated that the nucleotide sequences positionedupstream of these 90 types of genes and represented by SEQ ID NO: 1 to90 serve as stress responsive promoters.

EXAMPLE 2. Isolation of Gene Encoding Environmental Stress ResponsiveTranscriptional Factor

1. Materials and Methods

(1) Arabidopsis cDNA Clone

A microarray was constructed by using about 7,000 cDNA molecules intotal including genes isolated from Arabidopsis full-length cDNAlibraries, responsive to dehydration (RD) genes, early responsive todehydration (ERD) genes, kin 1 genes, kin2 genes, and cor15a genes;fragments amplified from λ control template DNA by PCR as an internalstandard; and mouse nicotinic acetylcholine receptor epsilon subunit(nAChRE) genes and mouse glucocorticoid receptor homologous genes, asnegative controls.

-   Positive control: dehydration-inducible genes (responsive to    dehydration genes: rd, and early responsive to dehydration genes:    erd);-   Internal standard: fragments amplified from λ control template DNA    by PCR (TX803, manufactured by Takara Shuzo, hereinafter referred to    as a “control fragment”);-   Negative control: mouse nicotinic acetylcholine receptor epsilon    subunit (nAChRE) genes and mouse glucocorticoid receptor homologous    genes, as negative controls, which do not substantially have    homology with any given sequence in an Arabidopsis database for    analyzing non-specific hybridization.    (2) Arabidopsis Full-Length cDNA Microarray

The present inventors have constructed full-length cDNA libraries froman Arabidopsis plant body under different conditions (e.g., dehydrationtreatment, cold treatment and non-treatment in different growth stagesfrom budding to maturation of seeds) by the biotinylated CAP trappermethod. From the full-length cDNA libraries, the present inventorsisolated individually about 7,000 independent Arabidopsis full-lengthcDNA molecules. The cDNA fragments, which were amplified by PCR, werearranged on a slide glass in accordance with a known method (Eisen andBrown, 1999). The present inventors prepared a full-length cDNAmicroarray containing about 7,000 Arabidopsis full-length cDNAmolecules, which contain the genes below.

(3) Dehydration-, Cold-, and High Salt-Inducible Genes Using cDNAMicroarray

In this example, dehydration-, cold- and high salt-inducible genes wereisolated by using a full length cDNA microarray containing about 7,000Arabidopsis full-length cDNA molecules.

Probes of plants treated with different stresses and an untreated plantwith stress and labeled with Cy3 and Cy5 fluorescent dyes were mixed.The probes were hybridized with the full-length cDNA microarraycontaining about 7,000 Arabidopsis full-length cDNA molecules. By such adouble labeling of a pair of cDNA probes wherein one of the mRNA sampleswas labeled with Cy3-dUTP and the other was labeled with Cy5-dUTP,hybridization with DNA elements on a microarray can be performedsimultaneously, with the result that quantitative determination of geneexpression under two different conditions (that is, stressed andunstressed conditions) can be directly and easily performed. Thehybridized microarray was scanned by two discrete laser channels for Cy3and Cy5 emission from each of DNA elements. Subsequently, the intensityratio between two fluorescent signals from each DNA element wasdetermined. Based on the relative value of the intensity ratio, a changeof differential expression of genes represented as a cDNA spot on themicroarray was determined. In this example, an α-tubulin gene, whoseexpression level was almost equivalent under two different experimentalconditions, was used as an internal control gene.

In the full-length cDNA microarray containing about 7,000 Arabidopsisfull-length cDNA molecules, a procedure for identifying dehydration-,cold-, and high salt-inducible genes will be explained.

Both mRNA molecules derived from a plant treated with one of thestresses mentioned above and mRNA molecules derived from a wild-typeplant unstressed were used to prepare Cy3-labeled cDNA and Cy5-labeledcDNA probes, respectively. These cDNA probes were mixed and hybridizedwith a cDNA microarray. In this example, a control fragment, whichexhibits almost the same expression level under two type conditions, wasused as an internal control gene. A gene that exhibits the expressionratio (dehydration/unstressed, cold/unstressed or high salt/unstressed)more than 5 times of that of the control fragment was defined as aninducible gene by a stress given to the gene.

(4) Analysis of Sequence

Plasmid DNA extracted by a DNA extraction device (model Biomek,manufactured by Beckman Coulter) and purified by use of a multiscreen96-hole filter plate (manufactured by Millipore) was sequenced to findhomology of gene sequences. A DNA sequence was determined by a dyeterminator cycle sequencing method using a DNA sequencer (ABI PRISM3700. PE Applied Biosystems, CA, USA). Based on the GenBank/EMBLdatabase, the homology of sequences was found by using the BLASTprogram.

(5) Amplification of cDNA

λZAP and λ FLC-1 were used as a vector for constructing a cDNA library.The cDNA inserted in a vector for the library was amplified by PCR usingcomplementary primers to the sequences of both sides of the cDNA.

The sequences of primers are as follows: FL forward 1224:5′-CGCCAGGGTTTTCCCAGTCACGA (SEQ ID NO: 165) FL reverse 1233:5′-AGCGGATAACAATTTCACACAGGA (SEQ ID NO: 166)

To 100 μl of a PCR solution mixture (0.25 mM dNTP, 0.2 μM PCR primer,1×Ex Taq Buffer, and 1.25 U of Ex Taq polymerase (manufactured by TakaraShuzo)), a plasmid (1 to 2 ng) was added as a template. PCR wasperformed under the following conditions: initial reaction at 94° C. for3 minutes, 35 cycles each consisting of 95° C. for one minute, 60° C.for 30 seconds, and 72° C. for 3 minutes, and a final reaction at 72° C.for 3 minutes. After a PCR product was precipitated with ethanol, theprecipitate was dissolved in 25 μl of 3×SSC and subjected toelectrophoresis using 0.7% agarose gel. The quality of the DNA obtainedand amplification efficiency of PCR were conformed.

(6) Construction of cDNA Microarray

Using a gene tip microarray stamp machine GTMASS SYSTEM (manufactured byNippon Laser & Electronics Lab.), 0.5 μl of a PCR product (500-1,000ng/ml) was loaded from a 384-well microtiter plate and form spots of thePCR product (5 nl for each) at intervals of 300 μm on 48 micro slideglasses (model Super Aldehyde substrate, manufactured by TelechemInternational). After spotting, the slide was dried in an atmospherehaving a relative humidity of 30% or less and irradiated withultraviolet rays for mediating a cross-linking reaction.

Thereafter, the slide was treated in 0.2% SDS with shaking for 2 minutesthree times and soaked in distilled water twice. Subsequently, theslides were placed on a slide rack, which was the transferred into achamber containing hot water and allowed to stand for 2 minutes.Subsequently, to the chamber, a blocking solution (containing 1 gborohydride, 300 ml of PBS, and 90 ml of 100% ethanol) was poured. Afterthe glass chamber housing the slide rack was moderately shaken, theslide rack was transferred to a chamber containing 0.2% SDS and gentlyshaken for one minute 3 times. Thereafter, the slide rack wastransferred to a glass chamber containing distilled water, moderatelyshaken for one minute, and centrifuged for 20 minutes to dry.

(7) Plant Material and Isolation of RNA

As a plant material, use was made of a wild type Arabidopsis thalianaplant body which was seeded on an agar medium and grown for 3 weeks(Yamaguchi-Shinozaki and Shinozaki, 1994) and an Arabidopsis thaliana(Colombian species) plant body into which DREB1A cDNA (Kasuga et al.,1999) connected to a 35S promoter of a cauliflower mosaic virus wasintroduced. Dehydration- and cold-stress treatments were performed inaccordance with the method of Yamaguchi-Shinozaki and Shinozaki (1994).More specifically, dehydration treatment was performed by pulling aplant body out of the agar medium, placing it on a filter, and dried ata temperature of 22° C. and a relative humidity of 60%. The coldtreatment was performed by transferring a plant body grown at 22° C. to4° C. High salt stress treatment was perfonned by growing a plant bodyat an aqueous solution containing 250 mM NaCl.

After wild type plant bodies were exposed to stress-treatment for 2 or10 hours, a sample was taken from each of plant bodies and stored incryogenic conditions with liquid nitrogen. Furthermore, wild type andDREB1A overexpresstion-type transformants cultured in an agar mediumwithout kanamycin were subjected to an experiment for identifying aDREB1A target gene. The DREB1A overexpresstion-type transformant was nottreated with stresses. The total RNA was isolated from the plant body byusing ISOGEN (Nippon gene, Tokyo, Japan) and mRNA was isolated andpurified by Oligotex-dT30 mRNA purification kit (Takara, Tokyo, Japan).

(8) Fluorescent Labeling of Probe

Each of the mRNA samples was subjected to a reverse transcriptionreaction in the presence of Cy3 dUTP or Cy5 dUTP (Amersham Pharmacia).More specifically, the reverse transcription reaction was performed in atotal amount of 20 μl of 1× Superscript first-stand buffer (containing50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, and 20 mM DTT,manufactured by Life Technology), which contained:

-   1 μg of denatured poly (A)⁺ which contains 1 ng of λ poly A⁺RNA-A    (TX802, manufactured by Takara Shuzo) serving as an internal    standard;-   50 ng/μl 12 to 18 mer oligo dT primer (manufactured by Life    Technology);-   0.5 mM dATP, 0.5 mM dGDP, 0.5 mM dCTP, and 0.2 mM dTTP;-   0.1 mM Cy3 dUTP or Cy5 dUTP;-   100 U of Rnase inhibitor;-   10 mM DTT; and-   200 U of Superscript II reverse transcriptase.

After the reaction solution of the aforementioned composition wasincubated at 42° C. for 35 minutes, 200 U of Superscript II reversetranscriptase was added and further incubated at 42° C. for 35 minutes.To this reaction mixture, subsequently, 5 μl of 0.5 M EDTA, 10 μl of 1NNaOH, and 20 μl of distilled water were added, thereby terminating theenzyme reaction taking place in the reaction solution and simultaneouslydecomposing a template. The reaction solution was then incubated at 65°C. for 1 hour and thereafter neutralized with 1M Tris-HCL (pH 7.5).

The reaction solution was transferred to a Microcon 30 microconcentrator (manufactured by Amicon). 250 μl of TE buffer was added andcentrifuged until the amount of the buffer reached 10 μl. The effulentwas discarded. This step was repeated 4 times. Probes contained in thereaction solution were centrifugally collected and several μl ofdistilled water was added. To the obtained probes, 5.1 μl of 20×SSC, 2μg/μl of Yeast tRNA, and 4.8 μl of 2% SDS were added. Further, thesamples were denatured at 100° C. for 2 minutes, placed at roomtemperature for 5 minutes, and thereafter used in hybridization.

(9) Microarray Hybridization and Scanning

A probe was centrifuged for one minute by a benchtop micro centrifuge.To avoid generation of bubbles, the probe was placed at the center of anarray and a cover slip was placed thereon. Four drops of 5 μl of 3×SSCwere dropped on a slide glass and a chamber was kept at a suitablehumidity to prevent the probe from being dried during hybridization.After the slide glass was placed in a cassette for hybridization (THC-1,BM machine) and the cassette was sealed, hybridization treatment wasperformed at 65° C. for 12 to 16 hours. The slide glass was taken outfrom the cassette and placed on the slide rack. After the cover slip wascarefully removed in solution 1 (2×SSC, 0.03% SDS), the rack was washedwhile shaking and transferred into solution 2 (1×SSC) to wash for 2minutes. The rack was further transferred to solution 3 (0.05×SSC),allowed to stand for 2 minutes, and centrifuged at 2500 g for 1 min todry.

The microarray was scanned at a resolution of 10 μm per pixel by use ofa scanning laser microscope (ScanArray 4000; GSI Lumonics, Watertown,Mass.). As a program for analyzing microarray data, QuantArray, Ver 2.0(GSI Lumonics) was used. The background fluorescence was obtainedthrough calculation based on fluorescent signals obtained from negativecontrol genes (mouse nicotinic acetylcholine receptor epsilon subunit(nAChRE) gene and mouse glucocorticoid receptor homologous gene).Samples giving a fluorescent signal value of less than 1,000, which isequal to less than twice the background signal value, were not subjectedto analysis. The cluster analysis of genes was performed by Genespring(manufactured by Silicon Genetic).

(10) Northern Analysis

Northern analysis was performed using total RNA, (Yamaguchi-Shinozakiand Shinozaki, 1994). DNA fragments were isolated from an Arabidopsisthaliana full-length cDNA library by a PCR method and used as probes forNorthern hybridization.

(11) Determination of Gene Encoding Transcriptional Factor

Based on the genomic information of Arabidopsis thaliana in a data base(GenBank/EMBL, ABRC), a gene encoding transcriptional factor wasanalyzed by using the BLAST program for gene analysis.

2. Results

(1) Identification of Stress-Inducible Gene

Fluorescence-labeled cDNA was prepared by subjecting mRNA isolated fromunstressed Arabidopsis thaliana to a reverse transcription reaction inthe presence of Cy5-dUTP. A second probe labeled with Cy3-dUTP wasprepared from a plant stress with dehydration, cold or high-salt. Bothprobes were simultaneously hybridized with a cDNA microarray containingabout 7,000 Arabidopsis thaliana cDNA clones and pseudo color image wascreated.

Genes induced and suppressed by a stress are represented by a red signaland a green signal, respectively. Genes expressed at virtually the samelevel in both treatments are represented by a yellow signal. Theintensity of each spot corresponds to the absolute value of theexpression level of each gene. It is shown that a cold-inducible gene(rd29A) is represented by a red signal, whereas a control fragment (aninternal control) is represented by a yellow signal.

As a result of scanning the microarray, 277 genes induced by dehydrationtreatment, 53 genes induced by cold treatment, and 194 genes induced byhigh salt treatment were identified. Note that genes whose expressionratio are not less than 5 times as large as that of a control fragmentwere determined as ones induced by a variety of stresses.

As a result of analysis using a database, 35 transcriptional factors,which were classified into the following families were identified. Notethat RAFL05-21-L12 was not classified into the following families.However, when the nucleic acid base sequence, which was searched by theBLAST X based on amino acid sequence data registered in the GenBankDatabase, it exhibited E-value of e⁻¹⁰⁰, which means that RAFL05-21-L12is homologous to a gene encoding a known transcriptional factor, thatis, heat shock transcriptional factor-like protein. As a result,RAFL05-21-L12 was identified as a transcriptional factor. In conclusion,36 types of transcriptional factors were identified in this example.

-   (1) DREB family: RAFL05-11-M11, RAFL06-11-K21, RAFL05-16-H23,    RAFL08-16-D06;-   (2) ERF family: RAFL08-16-G17, RAFL06-08-H20;-   (3) Zinc finger family: RAFL07-10-G04, RAFL04-17-D16, RAFL05-19-M20,    RAFL08-11-M13, RAFL04-15-K19, RAFL05-11-L01, RAFL05-14-C11,    RAFL05-19-G24, RAFL05-20-N02;-   (4) WRKY family: RAFL05-18-H12, RAFL05-19-E19, RAFL06-10-D22,    RAFL06-12-M01;-   (5) MYB family: RAFL05-14-D24, RAFL05-20-N17, RAFL04-17-F21;-   (6) bHLH family: RAFL09-12-N16;-   (7) NAC family: RAFL05-19-I05, RAFL05-21-I22, RAFL08-11-H20,    RAFL05-21-C17, RAFL05-08-D06;-   (8) Homeo domain family: RAFL05-20-M16, RAFL11-01-J18;    RAFL11-09-C20; and-   (9) bZIP family: RAFL05-18-N16, RAFL11-10-D10, RAFL04-17-N22,    RAFL05-09-G15.    (3) The Relationship between Treatment Time with Each Stress and    Expression Ratio

Genes encoding 36 types of stress responsive transcriptional factorsisolated as described above were analyzed for the relationship betweentreatment time with each stress and expression ratio. The results areshown in FIGS. 106 to 162. The correspondence between the names of genesand stress treatment shown in FIGS. 106 to 162 is listed in Table 5.TABLE 5 Number of figure Name of gene Type of stress RAFL08-16-G17 Highlevel salt solution RAFL05-11-M11 Dehydration RAFL05-11-M11 High levelsalt solution RAFL06-11-K21 High level salt solution RAFL06-11-K21Dehydration RAFL06-08-H20 Dehydration RAFL06-08-H20 High level saltsolution RAFL05-16-H23 High level salt solution RAFL05-16-H23Dehydration RAFL08-16-D06 Dehydration RAFL07-10-G04 DehydrationRAFL04-17-D16 Dehydration RAFL05-19-M20 Dehydration RAFL08-11-M13 Highlevel salt solution RAFL04-15-K19 Dehydration RAFL04-15-K19 ColdRAFL05-11-L01 Dehydration RAFL05-11-L01 High level salt solutionRAFL05-14-C11 Dehydration RAFL05-19-G24 High level salt solutionRAFL05-19-G24 Dehydration RAFL05-19-G24 Cold RAFL05-20-N02 DehydrationRAFL05-18-H12 Dehydration RAFL05-18-H12 High level salt solutionRAFL05-19-E19 High level salt solution RAFL06-10-D22 High level saltsolution RAFL06-12-M01 High level salt solution RAFL06-12-M01Dehydration RAFL05-14-D24 Dehydration RAFL05-14-D24 High level saltsolution RAFL05-20-N17 Cold RAFL05-20-N17 Dehydration RAFL04-17-F21Dehydration RAFL09-12-N16 Dehydration RAFL05-19-I05 DehydrationRAFL05-19-I05 High level salt solution RAFL05-21-I22 High level saltsolution RAFL08-11-H20 Dehydration RAFL08-11-H20 High level saltsolution RAFL05-21-C17 High level salt solution RAFL05-21-C17Dehydration RAFL05-08-D06 High level salt solution RAFL05-20-M16Dehydration RAFL05-20-M16 High level salt solution RAFL11-01-J18Dehydration RAFL11-01-J18 High level salt solution RAFL11-09-C20 Highlevel salt solution RAFL05-18-N16 High level salt solution RAFL11-10-D10Dehydration RAFL11-10-D10 High level salt solution RAFL04-17-N22Dehydration RAFL04-17-N22 High level salt solution RAFL05-09-G15Dehydration RAFL05-09-G15 High level salt solution RAFL05-21-L12Dehydration RAFL05-21-L12 High level salt solution

In FIGS. 106 to 162, the vertical axis shows the expression ratio of agene, which is calculated as follows:Expression ratio=[(FI of cDNA molecule under stress)/(FI of cDNAmolecule under no stress)]÷[(FI of control fragment under stress)/(FI ofcontrol fragment under no stress)]where FI is the intensity of fluorescence.

As shown in FIGS. 106 to 162, the genes encoding stress responsivetranscriptional factors isolated by a method according to the presentinvention exhibit different profiles; however, it is found thatexpression is induced by adding each stress.

INDUSTRIAL APPLICABILITY

A stress responsive promoter and an environmental stress responsivetranscriptional factor are provided by the present invention. Thepromoter of the present invention is useful in that it can be used forbreeding of environmental stress resistant plants in a molecular level.

Sequencing Free Text

SEQ ID NOS: 91, 92, 165 and 166 are synthetic primers.

1. An environmental stress-responsive promoter, comprising: (a) DNAconsisting of any nucleotide sequence selected from SEQ ID NOS: 1 to 90;(b) DNA consisting of a nucleotide sequence comprising a deletion,substitution or addition of one or more nucleotides relative to anynucleotide sequence selected from SEQ ID NOS: 1 to 90, and functioningas an environmental stress responsive promoter; and (c) DNA hybridizingunder stringent conditions to DNA consisting of any nucleotide sequenceselected from SEQ ID NOS: 1 to 90, and functioning as an environmentalstress responsive promoter.
 2. The promoter according to claim 1,wherein the environmental stress is at least one selected from the groupconsisting of cold stress, drought stress, and salt stress.
 3. Anexpression vector comprising a promoter according either one of claims 1and
 2. 4. An expression vector according to claim 3, further comprisingan arbitrary gene incorporated therein.
 5. A transformant comprising theexpression vector according to claim
 3. 6. A transgenic plant comprisingthe expression vector according to claim
 3. 7. The transgenic plantaccording to claim 6, wherein the plant is a plant body, plant organ,plant tissue or plant culture cell.
 8. A method for producing astress-resistant plant, comprising culturing or cultivating thetransgenic plant according to claim
 6. 9. A gene encoding anenvironmental stress-responsive transcriptional factor comprising: (a)any amino acid sequence selected from SEQ ID NOS: 2n (n is an integerfrom 47 to 82); (b) an amino acid sequence comprising a deletion,substitution or addition of one or more amino acids relative to anyamino acid sequence selected from SEQ ID NOS: 2n (n is an integer from47 to 82), functioning as an environmental stress responsive promoter,and having a transcriptional-factor activating activity.
 10. The geneaccording to claim 1, comprising: (a) DNA consisting of any nucleotidesequence selected from SEQ ID NOS: 2n−1 (n is an integer from 47 to 82);(b) DNA consisting of a nucleotide sequence comprising a deletion,substitution or addition of one or more nucleotides relative to anynucleotide sequence selected from SEQ ID NOS: 2n−1 (n is an integer from47 to 82), and encoding an environmental stress responsive promoter; and(c) DNA hybridizing under stringent conditions to DNA consisting of anynucleotide sequence selected from SEQ ID NOS: 2n−1 (n is an integer from47 to 82), and encoding an environmental stress responsive promoter. 11.The gene according to claim 9 or 10, wherein the environmental stress isat least one selected from the group consisting of cold stress, droughtstress, and salt stress.
 12. An expression vector comprising a geneaccording to claim 9 or
 10. 13. A transformant comprising the expressionvector according to claim
 12. 14. A transgenic plant comprising theexpression vector according to claim
 12. 15. The transgenic plantaccording to claim 14, wherein the plant is a plant body, plant organ,plant tissue or plant culture cell.
 16. A method for producing astress-resistant plant, comprising culturing or cultivating thetransgenic plant according to claim 14.